The present invention relates to methods and compositions for inhibiting metastatic spread of cancer and/or inhibiting progression of pre-existing metastatic disease in a subject using l1cam inhibition.
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9. A method of inhibiting progression of metastatic disease in a subject who has received treatment for the primary cancer, comprising administering to the subject a therapeutic amount of a l1cam inhibitor, wherein the l1cam inhibitor is administered:
(a) at least one month after a course of chemotherapy, targeted therapy, immunotherapy and/or radiotherapy of the primary cancer is completed;
(b) after the subject has achieved remission of the primary cancer; or
(c) after a surgical excision of the primary cancer or a metastasis has been completed.
8. A method of inhibiting metastatic spread of a primary cancer in a subject who has received treatment for the primary cancer, comprising administering to the subject a therapeutic amount of a l1cam inhibitor, wherein the l1cam inhibitor is administered:
(a) at least one month after a course of chemotherapy, targeted therapy, immunotherapy and/or radiotherapy of the primary cancer is completed;
(b) after the subject has achieved remission of the primary cancer; or
(c) after a surgical excision of the primary cancer or a metastasis has been completed.
1. A method of reducing the risk, in a subject who has received treatment for a primary cancer, of metastatic spread of the primary cancer, comprising administering to the subject a therapeutic amount of a l1cam inhibitor, wherein the l1cam inhibitor is administered:
(a) at least one month after a course of chemotherapy, targeted therapy, immunotherapy and/or radiotherapy of the primary cancer is completed;
(b) after the subject has achieved remission of the primary cancer; or
(c) after a surgical excision of the primary cancer or a metastasis has been completed.
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This application is a Continuation of International Patent Application No. PCT/US2017/045145, filed Aug. 2, 2017, which claims priority to U.S. Provisional Application No. 62/370,108 filed Aug. 2, 2016, the contents of each of which are hereby incorporated by reference in their entireties herein, and to each of which priority is claimed.
This invention was made with government support under Grant Nos. CA163167 awarded by the National Institutes of Health and W81XWH-12-1-0074 awarded by the United States Army Medical Research and Materiel Command. The government has certain rights in the invention.
The specification further incorporates by reference the Sequence Listing submitted herewith via EFS on Jan. 31, 2019. Pursuant to 37 C.F.R. § 1.52(e)(5), the Sequence Listing text file, identified as 0727340822SL.txt, is 1,434 bytes and was created on Jan. 31, 2019. The Sequence Listing, electronically filed herewith, does not extend beyond the scope of the specification and thus does not contain new matter.
The present invention relates to methods and compositions for inhibiting metastatic spread of cancer and/or inhibiting progression of pre-existing metastatic disease in a subject In particular embodiments, it provides for methods comprising treating the subject with an L1CAM inhibitor using a regimen that targets slow-growing metastatic cancer stem-like cells (“MetCSCs”) as exist, for example, in post-chemotherapy residual disease. It further provides for models of metastatic disease comprising MetCSC-expressing L1CAM that may be used to study metastatic progression of cancer and to identify useful therapeutic agents.
Despite recent advances in cancer therapeutics, metastasis remains the main cause of cancer death. Chemotherapy and targeted therapies for metastatic disease may induce tumor responses, but are nearly always followed by resistance and lethal relapse. The residual disease that persists after therapy and drives regrowth has been proposed to contain metastatic cancer stem-like cells (MetCSCs) that are particularly capable of self-renewal, and that are slow cell-cycling, tumor re-initiating and therapy resistant (Oskarsson et al., 2014; Hanahan et al., 2011; Malladi et al., 2016). Targeting MetCSCs may offer an important approach for treating metastatic cancer and micrometastatic residual disease in the adjuvant setting.
L1CAM was originally identified as a neuronal adhesion molecule (Rathjen et al., 1984; Maness and Schachner, 2007). L1CAM is a large, multidomain protein ectopically expressed at the invasion fronts of many solid tumors and universally associated with metastasis and poor prognosis (e.g., Altevogt et al., 2015). Metastatic lung and breast cancer single cells invading the brain use L1CAM to intimately stretch along blood vessels, in a process termed vascular co-option (Valiente et al., 2014; PCT/US2014/056379). RNAi-mediated L1CAM knockdown inhibits vascular co-option and prevents the outgrowth of brain macrometastases (PCT/US2014/056379).
The present invention relates to methods of preventing and treating metastatic disease, assay systems for identifying therapeutic agents, and compositions useful therefor.
It is based, at least in part, on the discovery that L1CAM is a marker of MetCSCs, and is expressed on these quiescent, very slowly dividing cells that can therefore escape standard chemotherapy and later re-initiate tumor growth. It is further based on the discovery that L1CAM-depletion inhibits the initiation of metastasis not only in the brain, but also in the lungs, liver and bone from breast, lung, colon and renal cancer xenografts, demonstrating the importance of L1CAM in the initiation of multi-organ metastasis. In particular, inducible L1CAM knockdown in advanced macrometastatic xenografts was observed to inhibit the progression of metastases, highlighting the clinical relevance of L1CAM inhibition in established metastatic disease. It is further based, in part, on the discoveiesy that L1CAM inhibition inhibited the growth of chemoresistant lung cancer xenografts, supporting a distinct mechanism of action from cytotoxic agents, and that inhibition of L1CAM was observed to render chemoresistant tumor cells sensitive to chemotherapy.
For clarity of description, and not by way of limitation, the detailed description of the invention is divided into the following subsections:
(i) methods of treatment;
(ii) assay systems; and
(iii) kits.
In various non-limiting embodiments, the present invention provides for a method of reducing the risk, in a subject who has received or is receiving treatment for a primary cancer, of metastatic spread of the primary cancer, comprising administering to the subject a therapeutic amount of a L1CAM inhibitor. In certain non-limiting embodiments, the L1CAM inhibitor is administered after one or more cycle of chemotherapy, targeted therapy, and/or immunotherapy of the primary cancer has been completed. In certain non-limiting embodiments, the L1CAM inhibitor is administered after a radiotherapy regimen of the primary cancer has been completed. In certain non-limiting embodiments, the L1CAM inhibitor is administered after an essentially complete (no cancer in the margins) surgical excision of the primary cancer or a metastasis has been completed. In certain non-limiting embodiments, the L1CAM inhibitor is administered in a maintenance regimen (e.g., administered at regular intervals (e.g., at least once a week, at least once a month, at least once every two months, at least once every three months, at least once every six months)) for a period of time after a cycle of treatment, or surgical excision, of the primary cancer. The period of time for maintenance therapy may be at least about three months or at least about 6 months or at least about one year or at least about 2 years. In certain non-limiting embodiments, the L1CAM inhibitor is administered after the subject has achieved remission of the primary cancer. In certain non-limiting embodiments, the L1CAM inhibitor is administered in a maintenance regimen (e.g., administered at regular intervals (e.g., at least once a week, at least once a month, at least once every two months, at least once every three months, at least once every six months)) for a period of time after achieving remission of the primary cancer. The period of time for maintenance therapy may be at least about three months or at least about 6 months or at least about one year or at least about 2 years.
In various non-limiting embodiments, the present invention provides for a method of inhibiting metastatic spread of a primary cancer in a subject who has received treatment for the primary cancer, comprising administering to the subject a therapeutic amount of a L1CAM inhibitor. In certain non-limiting embodiments, the L1CAM inhibitor is administered after one or more cycle of chemotherapy, targeted therapy, and/or immunotherapy of the primary cancer has been completed. In certain non-limiting embodiments, the L1CAM inhibitor is administered after a radiotherapy regimen of the primary cancer has been completed. In certain non-limiting embodiments, the L1CAM inhibitor is administered after an essentially complete (no cancer in the margins) surgical excision of the primary cancer or a metastasis has been completed. In certain non-limiting embodiments, the L1CAM inhibitor is administered in a maintenance regimen (e.g., administered at regular intervals (e.g., at least once a week, at least once a month, at least once every two months, at least once every three months, at least once every six months)) for a period of time after a cycle of treatment, or surgical excision, of the primary cancer. The period of time for maintenance therapy may be at least about three months or at least about 6 months or at least about one year or at least about 2 years. In certain non-limiting embodiments, the L1CAM inhibitor is administered after the subject has achieved remission of the primary cancer. In certain non-limiting embodiments, the L1CAM inhibitor is administered in a maintenance regimen (e.g., administered at regular intervals (e.g., at least once a week, at least once a month, at least once every two months, at least once every three months, at least once every six months)) for a period of time after achieving remission of the primary cancer. The period of time for maintenance therapy may be at least about three months or at least about 6 months or at least about one year or at least about 2 years.
In various non-limiting embodiments, the present invention provides for a method of inhibiting progression of metastatic disease in a subject comprising administering to the subject a therapeutic amount of a L1CAM inhibitor. In various non-limiting embodiments, the present invention provides for a method of inhibiting progression of metastatic disease in a subject who has received treatment for the primary cancer, comprising administering to the subject a therapeutic amount of a L1CAM inhibitor. In certain non-limiting embodiments, the L1CAM inhibitor is administered after one or more cycle of chemotherapy, targeted therapy, and/or immunotherapy of the primary cancer has been completed. In certain non-limiting embodiments, the L1CAM inhibitor is administered after a radiotherapy regimen of the primary cancer has been completed. In certain non-limiting embodiments, the L1CAM inhibitor is administered after an essentially complete (no cancer in the margins) surgical excision of the primary cancer or a metastasis has been completed. In certain non-limiting embodiments, the L1CAM inhibitor is administered in a maintenance regimen (e.g., administered at regular intervals (e.g., at least once a week, at least once a month, at least once every two months, at least once every three months, at least once every six months)) for a period of time after a cycle of treatment, or surgical excision, of the primary cancer. The period of time for maintenance therapy may be at least about three months or at least about 6 months or at least about one year or at least about 2 years. In certain non-limiting embodiments, the L1CAM inhibitor is administered after the subject has achieved remission of the primary cancer. In certain non-limiting embodiments, the L1CAM inhibitor is administered in a maintenance regimen (e.g., administered at regular intervals (e.g., at least once a week, at least once a month, at least once every two months, at least once every three months, at least once every six months)) for a period of time after achieving remission of the primary cancer. The period of time for maintenance therapy may be at least about three months or at least about 6 months or at least about one year or at least about 2 years.
In various non-limiting embodiments, the present invention provides for a method of inhibiting progression of metastatic disease in a subject comprising administering to the subject a therapeutic amount of an agent that reduces L1CAM expression in cancer cells, for example via CRISPR/Cas9 mediated gene editing.
A metastasis is a population of cancer cells at a location that is not physically contiguous with the original location of the cancer.
“Reducing the risk of metastatic spread” is relative to the risk of metastatic spread in a comparable control subject not treated with an L1CAM inhibitor.
“Inhibiting metastatic spread of a primary cancer” means one or more of: reducing the number, location(s), and/or size, of metastasis/es, and/or increasing the period of time to occurrence of metastasis/es, and/or prolonging survival, relative to a comparable control subject not treated with an L1CAM inhibitor.
“Inhibiting progression of metastatic disease” means one or more of the following: decreasing the size of existing metastasis/es, reducing the rate of growth of existing metastasis/es, reducing the incidence of newly detectable metastasis/es, improving quality of life, and/or increasing time to recurrence, and/or prolonging survival, relative to a comparable control subject not treated with an L1CAM inhibitor.
In certain non-limiting embodiments, the invention provides, in a subject having a primary cancer, a method of inhibiting metastatic spread of the cancer, comprising determining whether a cell of the cancer expresses L1CAM and, if the cell does express L1CAM, administering to the subject, in addition to therapy of the primary cancer, a therapeutic amount of a L1CAM inhibitor.
In non-limiting embodiments, a further indicator of increased risk is high/medium surface expression of EphB2.
In various non-limiting embodiments, the subject is a human or a non-human animal, for example a dog, a cat, a horse, a rodent, a mouse, a rat, a hamster, a non-human primate, a rabbit, a sheep, a cow, a cetacean, etc.
In various non-limiting embodiments, the cancer is a breast cancer, a lung cancer, a renal cancer, a colorectal cancer, an ovarian cancer, a prostate cancer, a liver cancer, or a melanoma.
The site of metastasis may be, for example but not by way of limitation, brain, lung, bone, or liver.
In various non-limiting embodiments of the invention, an L1CAM inhibitor may be administered concurrently with a chemotherapy and/or targeted therapy and/or immunotherapy and/or radiotherapy regimen. However, in alternative non-limiting embodiments, an L1CAM inhibitor may be administered after a course of chemotherapy, targeted therapy, immunotherapy and/or radiotherapy is complete. In specific, non-limiting examples, the L1CAM inhibitor may be administered at the conclusion of the treatment regimen, or at least one month thereafter, or at least three months thereafter, or at least six months thereafter, or at least one year thereafter. In related non-limiting embodiments, an L1CAM inhibitor may be administered after an essentially complete (no cancer in the margins) surgical excision of the primary cancer or metastasis has been completed.
In various non-limiting embodiments, an L1CAM inhibitor may be administered in a maintenance regimen (e.g., administered at regular intervals (e.g., at least once a week, at least once a month, at least once every two months, at least once every three months, at least once every six months) for a period of time after a course of chemotherapy or radiation therapy is complete or after achieving complete or partial remission of the primary cancer. Said maintenance regimen may be followed whether or not active disease is determined to be present.
In various embodiments of the invention, a decision to use L1CAM inhibition as a treatment may be supported by determining that the cancer and/or its metastasis to be treated expresses L1CAM and optionally one or more of EphB2, CD133 and/or CD44. Expression of L1CAM may be determined by any method known in the art, for example as discussed in the sections below. In certain non-limiting embodiments, expression of L1CAM may be detected using an antibody specific for L1CAM or amplification of L1CAM-encoding mRNA using polymerase chain reaction (PCR).
In various non-limiting embodiments, the invention provides for a method of reversing chemoresistance of a cancer cell to a chemotherapy agent, comprising administering, to the cancer cell, an effective amount of L1CAM inhibitor. In non-limiting embodiments, the cancer cell is a metastatic cancer. In non-limiting embodiments, the cancer is a breast, lung, renal, or colorectal cancer. In non-limiting embodiments, the chemotherapeutic agent is carboplatin or methotrexate. In a specific non-limiting embodiment, the cancer is Kras-mutant lung cancer and the chemotherapeutic agent is carboplatin or methotrexate. “Reversing chemoresistance” means that administration of L1CAM inhibitor increases the sensitivity of the cancer cell, cells or tumor to the anti-cancer effect of the chemotherapeutic agent relative to a control cancer cell not treated with L1CAM inhibitor (for example, where the cancer cell is deemed to have a lower than expected response to the chemotherapeutic agent). In a specific non-limiting embodiment, the increase in sensitivity is at least about 30 percent.
An L1CAM inhibitor is an agent that reduces the ability of L1CAM to co-opt blood vessels and/or reduces the ability of L1CAM to reinitiate or promote tumor growth or spread (e.g., the inhibitor reduces tumor cell invasiveness) and can be used to eliminate quiescent cells within tumors. An L1CAM inhibitor may act, for example and not by way of limitation, by reducing expression of L1CAM in the cancer cell or removing L1CAM from the cancer cell surface or binding to L1CAM such that its ability to bind to an endothelial cell or other cancer cells or normal tissue is reduced, for example by reducing the amount of L1CAM available for cell binding, by physical inhibition or by labeling L1CAM-expressing cells and thus marking them for destruction by the immune system.
In non-limiting embodiments, where the subject is a human, L1CAM to be inhibited is human L1CAM having an amino acid sequence as set forth in UniProtKB Accession No. P32004 and/or NCBI Accession Nos. NM_000425 version NM_000425.4 and/or NM_001278116 version NM_001278116.1.
In non-limiting embodiments, an L1CAM inhibitor may be an immunoglobulin, for example an antibody or antibody fragment or single chain antibody that specifically binds to L1CAM, or a therapeutic molecule that comprises one or more immunoglobulin region(s). Non-limiting examples of such antibodies are disclosed in U.S. Pat. No. 8,138,313, International Patent Application Publication No. WO 2007114550, and International Patent Application Publication No. WO 2008151819, as well as antibodies that compete with the antibodies described in these citations for L1CAM binding. In certain non-limiting embodiments an anti-L1CAM antibody or antibody fragment may be used to prepare a human, humanized, or otherwise chimeric antibody that is specific for L1CAM for use according to the invention. In certain non-limiting embodiments, an L1CAM antibody, antibody fragment, or single chain antibody may inhibit binding of L1CAM to an endothelial cell or a blood capillary, to L1CAM or other molecules on neighboring cancer or stromal cells, or to other components of the extracellular matrix under physiologic conditions, for example in vitro or in vivo. In certain non-limiting embodiments, an L1CAM inhibitor comprises immunoglobulin regions that bind to L1CAM and CD133 (the immunoglobulin is bi-specific). In certain non-limiting embodiments, an L1CAM inhibitor comprises immunoglobulin regions that bind to L1CAM and CD44. In certain non-limiting embodiments, an L1CAM inhibitor binds to L1CAM as well as a T cell antigen. In certain non-limiting embodiments, an L1CAM inhibitor binds to L1CAM as well as an NK cell antigen. In certain non-limiting embodiments, an L1CAM inhibitor binds to L1CAM and EphB2.
In non-limiting embodiments, an L1CAM inhibitor may be a nucleic acid, for example, a short hairpin, interfering, antisense, or ribozyme nucleic acid comprising a region of homology to an L1CAM mRNA. For example, such nucleic acids may be between about 15 and 50 or between about 15 and 30 or between about 20 and 30 nucleotides long, and be able to hybridize to L1CAM mRNA under physiologic conditions. A non-limiting example of a short hairpin (sh) RNA that inhibits L1CAM is set forth in the example below. In non-limiting embodiments, an L1CAM inhibitor which is a nucleic acid may be provided in a L1CAM-expressing cancer cell via a vector, for example a lentivirus, which may be selectively targeted to said cancer cell and/or wherein expression of the L1CAM inhibitor nucleic acid may be directed by a promoter which is selectively active in tumor cells. Non-limiting examples of nucleic acid sequence of an L1CAM mRNA include the sequence set forth in NCBI Accession Nos. NM_000425 version NM_000425.4 and/or NM_001278116 version NM_001278116.1. In one specific non-limiting embodiment, the L1CAM inhibitor is RNAi TRCN0000063916 (The RNAi Consortium, Public TRC Portal), having a hairpin sequence
(SEQ ID NO: 1)
5′-CCGGACGGGCAACAACAGCAACTTTCTCGAGAAAGTTGCTGTTGTTG
CCCGTTTTTTG
and a target sequence ACGGGCAACAACAGCAACTTT (SEQ ID NO:2);
or the hairpin sequence
(SEQ ID NO: 3)
5′-CCGGCCACTTGTTTAAGGAGAGGATCTCGAGATCCTCTCCTTAAACA
AGTGGTTTTTG
and a target sequence CCACTTGTTTAAGGAGAGGAT (SEQ ID NO:4);
or the hairpin sequence
(SEQ ID NO: 5)
5′-CCGGGCCAATGCCTACATCTACGTTCTCGAGAACGTAGATGTAGGCA
TTGGCTTTTTG
and a target sequence GCCAATGCCTACATCTACGTT (SEQ ID NO:6)
In certain non-limiting embodiments, the L1CAM inhibitor may be an antibody directed against a mutated L1CAM protein that is expressed on the surface of a MetCSC at high level. Non-limiting examples of mutated L1CAM proteins are set forth in Vos, Y. J., and Hofstra, R. M. (2010) and in Faltas et al., 2016, Nat. Genet. 48(12): 1490-1499, both incorporated by reference herein. An updated and upgraded L1CAM mutation database. Hum Mutat 31, E1102-1109.
In certain non-limiting embodiments, the L1CAM inhibitor may be an agent or agents that can edit the L1CAM gene, for example via CRISPR/Cas9-mediated knockout of the L1CAM gene (see, e.g.
In various embodiments, the present invention relates to assay systems and components thereof for producing models of metastatic disease and using such models as assay systems for identifying therapeutic agents.
In various non-limiting embodiments, the invention provides for an assay for identifying an agent that inhibits metastasis, comprising an organoid culture comprising cancer cells that express L1CAM. Said cells may express high levels of L1CAM and/or medium or high levels of EphB2. In certain non-limiting embodiments, said cancer cells are MetCSCs and optionally express an exogenous marker, for example a fluorescent exogenous marker. Certain non-limiting embodiments provide for a method of identifying a MetCSC, comprising determining that the cell expresses L1CAM, for example surface expression of a high level of L1CAM. In certain non-limiting embodiments, the MetCSC further expresses a medium or high level of EphB2. Expression of L1CAM and optionally EphB2 may be determined by any method known in the art, including but not limited to antibody-based or PCR-based methods. In certain non-limiting embodiments, the MetCSC is isolated from a primary cancer of a subject. In certain non-limiting embodiments, the MetCSC is isolated from a metastasis of a subject. In certain non-limiting embodiments, the cancer, either primary or metastatic, is of breast, lung, renal, or colorectal origin.
In certain non-limiting embodiments, where the cancer, primary or metastatic, is of colorectal origin, the MetCSC may further be identified as exhibiting surface expression of one or more of CD133 and/or CD44 in addition to L1CAM and optionally EphB2.
Certain non-limiting embodiments provide for an isolated MetCSC cell expressing L1CAM. In certain non-limiting embodiments, the MetCSC expresses a high level of L1CAM. In certain non-limiting embodiments, the isolated MetCSC comprises an introduced exogenous marker. In certain non-limiting embodiments, the exogenous marker is a fluorescent marker. In particular embodiments, the invention provides for a composition comprising cells that are an essentially pure population of MetCSCs expressing L1CAM. In non-limiting embodiments, the MetCSCs may be isolated using FACS or other cell-isolating methods known in the art.
The above MetCSCs may be used to prepare a model system of metastasis that may be used to study the metastatic process and may be used as an assay system to identify agents for inhibiting and thereby treating metastatic disease in a subject.
In certain non-limiting embodiments, the invention provides for a model system of metastasis/assay system comprising an organoid culture formed of cancer cells that express L1CAM and optionally EPCAM. In certain non-limiting embodiments, the cancer cells further express EphB2. In certain embodiments the cancer cells express high levels of L1CAM and med/high levels of EphB2.
In certain non-limiting embodiments, the invention provides for a model system of metastasis/assay system comprising an organoid culture formed of MetCSC cells that express L1CAM and EPCAM. In certain non-limiting embodiments, the MetCSC cells further express EphB2. In certain embodiments the MetCSC cells express high levels of L1CAM and med/high levels of EphB2. See, for example, Drost et al., 2016, Nature Protocols 11:347-358 for description of organoid culturing techniques. For example, the culture medium for organoid culture may comprise Wnt. MetCSC cells, as described above, may be used as the basis for organoid development. A non-limiting example of an in vitro assay system comprises said MetCSC, under conditions that promote organoid formation. An agent effective in inhibiting metastasis from forming and/or progressing may be identified as an agent that reduces the occurrence or growth of organoids in said system.
In certain non-limiting embodiments, a MetCSC, as described above, optionally having been cultivated to form an organoid, may be introduced into a laboratory animal such as an athymic mouse or other immunocompromised non-human host, and used to test whether an administered agent is effective at delaying, reducing the number, or inhibiting the growth or dispersal of metastatic growth resulting from said MetCSC, thereby identifying it as having antimetastasis therapeutic activity.
In related non-limiting embodiments, the invention provides for a method of identifying an agent that inhibits metastasis, comprising:
(i) providing an organoid culture comprising cancer cells that express L1CAM;
(ii) contacting the organoid culture with a test agent;
(iii) determining whether the level of L1CAM expression decreases in the test agent-contacted culture relative to a control organoid culture that has not been contacted with the test agent;
wherein a decrease in the level of L1CAM expression, interactions, and/or signaling in response to contacting with the test agent indicates that the test agent inhibits metastasis.
In certain non-limiting embodiments, the invention provides for a kit for determining whether a subject having a cancer is at increased risk for metastatic spread of the cancer, comprising means for determining whether a cell of the cancer expresses L1CAM, and, optionally, instructional material that indicates that expression, on a cancer cell, of L1CAM indicates that the subject may benefit from L1CAM inhibitor therapy.
In certain non-limiting embodiments, the invention provides for a kit for identifying an agent that inhibits metastasis, comprising (i) cancer cells that express L1CAM and (ii) means for determining the L1CAM expression level. In various non-limiting embodiments, the cancer cells can express high levels of L1CAM and optionally further express one or more of CD133, CD44 and/or EphB2. In certain non-limiting embodiments, the means for detecting L1CAM expression is an oligonucleotide probe that detectably binds to L1CAM. In certain non-limiting embodiments, the means for detecting L1CAM expression is a pair of primers that can be used in polymerase chain reaction to determine the L1CAM expression level. In certain non-limiting embodiments, the means for detecting L1CAM expression is an immunoglobulin that specifically binds to L1CAM.
Non-limiting examples of types of kits include, but are not limited to, arrays/microarrays, L1CAM-specific antibodies and beads, which may contain one or more primer, probe, antibody, or other detection reagent(s) for detecting L1CAM and optionally other markers set forth above (EphB2, CD133, CD44).
In non-limiting embodiments, the present invention provides for a kit for determining whether a subject having a cancer is at increased risk of having or developing a metastasis of the cancer, comprising a means for detecting the protein level (directly or via mRNA) of L1CAM and optionally EphB2, CD133 and/or CD44.
In certain non-limiting embodiments, surface expression of L1CAM and optionally EphB2, C44, and/or CD133 is detected.
In non-limiting embodiments, a kit may comprise at least one antibody for immunodetection of L1CAM and optionally EphB2, CD44 and/or CD133. Antibodies, both polyclonal and monoclonal, including molecules comprising an antibody variable region or subregion thereof, specific for these proteins, may be prepared using conventional immunization techniques, as will be generally known to those of skill in the art. The immunodetection reagents of the kit may include detectable labels that are associated with, or linked to, the given antibody or antigen itself. Such detectable labels include, for example, chemiluminescent or fluorescent molecules (rhodamine, fluorescein, green fluorescent protein, luciferase, Cy3, Cy5, or ROX), radiolabels (3H, 35S, 32P, 14C, 1311) or enzymes (alkaline phosphatase, horseradish peroxidase). Alternatively, a detectable moiety may be comprised in a secondary antibody or antibody fragment which selectively binds to the first antibody or antibody fragment (where said first antibody or antibody fragment specifically recognizes a serpin).
In a further non-limiting embodiment, a L1CAM-specific antibody (or optionally EphB2, CD44 and/or CD133-specific antibody) may be provided bound to a solid support, such as a column matrix, an array, or well of a microtiter plate. Alternatively, the support may be provided as a separate element of the kit.
In certain embodiments, types of kits include, but are not limited to, packaged probe and primer sets (e.g. TaqMan probe/primer sets), which may further contain one or more probes, primers, or other detection reagents for detecting one or more serpin, for example neuroserpin, serpin B2, serpin E1, serpin E2 or serpin D1.
In a specific, non-limiting embodiment, a kit may comprise a pair of oligonucleotide primers, suitable for polymerase chain reaction (PCR) or nucleic acid sequencing, for detecting the protein(s) to be identified. A pair of primers may comprise nucleotide sequences complementary L1CAM or optionally EphB2, CD133 and/or CD44-encoding mRNA and be of sufficient length to selectively hybridize with said mRNA. Multiple marker protein-specific primers may be included in the kit to simultaneously assay a plurality of proteins (e.g. L1CAM and optionally one or more of EphB2, CD44 and/or CD133). The kit may also comprise one or more polymerase, reverse transcriptase, and nucleotide bases, wherein the nucleotide bases can optionally be further detectably labeled.
In non-limiting embodiments, a primer may be at least about 10 nucleotides or at least about 15 nucleotides or at least about 20 nucleotides in length and/or up to about 200 nucleotides or up to about 150 nucleotides or up to about 100 nucleotides or up to about 75 nucleotides or up to about 50 nucleotides in length.
In a further non-limiting embodiment, an oligonucleotide primer may be immobilized on a solid surface or support, for example, on a nucleic acid microarray, and optionally the position of each oligonucleotide primer bound to the solid surface or support is known and identifiable.
In a specific, non-limiting embodiment, a kit may comprise at least one nucleic acid probe, suitable for in situ hybridization or fluorescent in situ hybridization, for detecting the protein to be identified.
In one specific non-limiting embodiment, a kit may comprise one or more of: a probe, primers, microarray, antibody or antibody fragment suitable for detecting L1CAM and one or more of EphB2, CD44, and/or CD133.
In certain non-limiting embodiments, a kit may comprise one or more detection reagents and other components (e.g. a buffer, enzymes such as alkaline phosphatase, antibodies, and the like) necessary to carry out an assay or reaction to determine the expression levels of a biomarker.
In certain non-limiting embodiments, the invention provides for a diagnostic method for determining whether a subject having a cancer is at increased risk for metastatic spread of the cancer, comprising means for determining whether a cell of the cancer expresses L1CAM and EphB2, where if the cancer cell is found to express L1CAM and EphB2, and particularly high L1CAM and med/high EphB2, the subject is at increased risk for developing metastatic disease relative to a subject having a cancer lacking those markers, and may benefit from L1CAM inhibitor therapy. The method may further include informing the subject or a health care worker of the result of the determination and the associated risk. The method may further include, where an increased risk is indicated, recommending or performing an additional diagnostic procedure, for example an imaging study, to determine whether the subject has detectable metastatic disease. Non-limiting examples of imaging modalities include magnetic resonance imaging, computerized tomography and positron emission tomography. In a related embodiment, the invention provides for a method of treatment comprising performing the diagnostic method and then, where increased risk is indicated, administering a therapeutic amount of L1CAM inhibitor.
Metastasis is a highly inefficient process, in that primary tumor cells must first undergo epitheial-mesenchymal transition and escape from the primary tumor. After dissemination in the bloodstream, the vast majority of tumor cells die, leaving only a tiny fraction capable of surviving in a hostile foreign organ. These remaining few tumor cells may lie dormant for months or years, and then, when conditions are right, start to proliferate and reinitiate tumor growth. Once this so-called macrometastatic growth has been initiated, it is usually still possible to kill the bulk of tumor cells with chemotherapy, radiation, targeted therapy and/or immunotherapy—sometimes even to the point of no measurable disease—but a true cure is rarely possible.
This suggests that the tumor cells that form macrometastases—the MetCSCs—are resistant to chemotherapy, radiation, targeted therapy and/or immunotherapy, as they must survive these therapies applied to treat, first, the primary tumor and, later, its metastases. In addition, MetCSCs are able to undergo long-term self-renewal, have the ability to generate heterogeneous progeny (recapitulating tumor heterogeneity) and are capable of entering and exiting a dormant state, in which they can, potentially, exist for years (even decades, as seen in the case of ER/PR positive breast cancer).
Because chemotherapy may not be a treatment option for controlling metastatic growth, it is important to understand MetCSC mechanistically and identify therapeutic targets that will specifically kill these cells. To date, model systems for studying metastases have been imperfect.
L1CAM is a molecule associated with various cancers. Aberrant L1CAM expression has been demonstrated at the leading edge of primary tumors, and is associated with invasion, metastasis and poor prognosis in many human cancers including lung, breast and colon carcinomas (Voura et al., 2001; Ben et al., 2010; Tsutsumi et al., 2011; Schroder et al, 2009; Tischler et al., 2011; Boo et al., 2007; Chen et al., 2013; Fogel et al., 2003a; Doberstein et al., 2011; Fogel et al., 2003b; Kim et al., 2009; Maness et al., 2007). L1CAM expression is normally restricted to neurons where it mediates axonal guidance through interactions of the growth cone with surrounding components (Castellani et al., 2002; Wiencken-Barger et al., 2004).
A number of immunohistologic studies were performed to further study the expression of L1CAM during the metastatic process. L1CAM was found to be expressed at the primary tumor invasion front (
Experiments were performed to determine whether inhibition of L1CAM could impact metastasis. Cancer cells which either were transfected with shL1CAM (to knock down L1CAM expression) or control cancer cells were introduced into athymic mice (by intracardiac injection to assess brain or bone metastasis and by tail vein injection to assess lung metastasis) and the amount of metastases determined after several weeks using bioluminescence imaging. As shown in
Experiments were also performed to determine whether L1CAM inhibition could be used to treat existing metastatic disease. In these experiments, expression of shL1CAM was placed under the control of an inducible promoter which could be activated by the drug doxycycline, so that knockdown of L1CAM could be turned on after dissemination of tumor cells had occurred. Athymic mice receiving either shL1CAMind-transfected cancer cells or control cancer cells were treated with doxycycline at day 14 and then assessed for metastatic disease by bioluminescence imaging at day 28 (
Experiments were performed to develop models for metastatic disease. In particular, patient-derived cells were used to generate organoids in culture, using a modification of the technique developed by Hans Clevers, in which organoids grow in three dimensions in matrigel and stem cell media enriched with Wnt is used for culturing (
It was further observed that tumor L1CAM expression was associated with organoid-initiating capability (
Interestingly, L1CAMhigh cells were found to give rise to both L1CAMhigh and L1CAMlow progeny (
When L1CAM expression was compared between dissociated tumor and the organoids generated from the dissociated tumor, it was found that L1CAM expression was a trait selected for during organoid generation. The results from dissociated tumor collected from two different patients are shown in
Patient metastasis-derived organoids were expanded in vitro, FACS sorted into L1CAMhigh and L1CAMlow populations and implanted as subcutaneous xenografts into NSG mice, whereupon the L1CAMhigh cells displayed greater in vivo tumor re-initiation capacity (
Next, we injected Stage III CRC-derived organoids into the splenic vein of immunocompromised NSG mice. Liver metastases thus generated were then passaged as organoids and re-injected into the splenic vein. Such serially passaged liver metastatic organoids not only formed larger liver metastases more rapidly than their parental organoids, but also expressed higher levels of L1CAM (
Kras-mutant lung cancer cells that were resistant to carboplatin and methotrexate were transfected with shCONTROL or shL1CAM operably linked to a doxycycline-inducible promoter. shCONTROL or shL1CAM containing cancer cells were then injected, intracardially, into athymic mice (day 0). On day 14, treatment with doxycycline and v=carboplatin or methotrexate was initiated, and tumors were assessed on day 35. As shown in
To interrogate whether L1CAM is functionally required for organoid growth or regeneration, we performed CRISPR-Cas9 mediated knockout of L1CAM in metastasis-derived organoids (see
Consistently, L1CAM-deficient cells displayed increased caspase activity in the first week following dissociation (
YAP activity is induced by loss of epithelial interaction and contact with stiff basement membrane in multiple contexts (Zhao B, Wei X, Li W, Udan R S, Yang Q, Kim J, Xie J, Ikenoue T, Yu J, Li L, Zheng P, Ye K, Chinnaiyan A, Halder G, Lai Z C, Guan K L. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 2007 Nov. 1; 21(21):2747-61, Aragona M, Panciera T, Manfrin A, Giulitti S, Michielin F, Elvassore N, Dupont S, Piccolo S. A mechanical checkpoint controls multicellular growth through YAP/TAZ regulation by actin-processing factors. Cell. 2013 Aug. 29; 154(5):1047-59. Benham-Pyle B W, Pruitt B L, Nelson W J. Cell adhesion. Mechanical strain induces E-cadherin-dependent Yapl and 3-catenin activation to drive cell cycle entry. Science. 2015 May 29; 348(6238): 1024-7. Gjorevski N, Sachs N, Manfrin A, Giger S, Bragina M E, Ordóñez-Morán P, Clevers H, Lutolf M P. Designer matrices for intestinal stem cell and organoid culture. Nature. 2016 Nov. 24; 539(7630):560-564). Indeed, dissociation of organoids into single cells significantly induced expression of YAP target genes ANKRD1, CYR61 and ITGB1 (
Since L1CAM is required for survival, regrowth and restoration of tissue architecture by transformed epithelial cells, we wondered whether it might also be required in non-transformed epithelia when epithelial integrity is disrupted. As seen in the human, normal mouse colon epithelia did not express significant amounts on L1CAM (
To interrogate the functional significance of L1CAM in colon regeneration, we crossed L1CAMfl/fl mice with the intestinal stem cell-specific Lgr5-GFP-IRES-Cre-ERT2 mice. Cre recombinase expression was induced by treating the mice with IP tamoxifen for three doses concurrent with DSS or water treatment. When given water, tamoxifen-treated mice displayed no alterations in weight (
Next, we sought to understand how epithelial progenitor cells induce and regulate L1CAM expression. To determine whether colitis-associated inflammatory cytokines might contribute to L1CAM induction, we incubated human CRC organoids with conditioned media from normal or inflamed colons. Neither colitis conditioned-media nor incubation with recombinant cytokines associated with colitis or neuronal regeneration (where L1CAM has been previously implicated) induced L1CAM (
In various non-neuronal tissues, the transcriptional repressor NSRF/REST normally prevents the expression of L1CAM and other neuronal genes. REST has been identified as a tumor suppressor and metastatic colorectal cancers frequently acquire loss-of-function mutations or deletions in the REST gene. We therefore investigated whether REST is functional in repressing L1CAM expression in organoids. REST knockdown in human CRC organoids strongly induced L1CAM expression, suggesting that REST is active in repressing L1CAM expression (
Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.
Massague, Joan, Ganesh, Karuna, Valiente, Manuel
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